Brain S100A5 Is a Novel Calcium-, Zinc-, and Copper Ion-binding Protein of the EF-hand Superfamily

Abstract

S100A5 is a novel member of the EF-hand superfamily of calcium-binding proteins that is poorly characterized at the protein level. Immunohistochemical analysis demonstrates that it is expressed in very restricted regions of the adult brain. Here we characterized the human recombinant S100A5, especially its interaction with Ca 2؉ , Zn 2؉ , and Cu 2؉. Flow dialysis revealed that the homodimeric S100A5 binds four Ca 2؉ ions with strong positive cooperativity and an affinity 20 -100-fold higher than the other S100 proteins studied under identical conditions. S100A5 also binds two Zn 2؉ ions and four Cu 2؉ ions per dimer. Cu 2؉ binding strongly impairs the binding of Ca 2؉ ; however, none of these ions change the ␣-helical-rich secondary structure. After covalent labeling of an exposed thiol with 2-(4-(iodoacetamide)anilino)-naphthalene-6-sulfonic acid, binding of Cu 2؉ , but not of Ca 2؉ or Zn 2؉ , strongly decreased its fluorescence. In light of the three-dimensional structure of S100 proteins, our data suggest that in each subunit the single Zn 2؉ site is located at the opposite side of the EF-hands. The two Cu 2؉ -binding sites likely share ligands of the EF-hands. The potential role of S100A5 in copper homeostasis is discussed.Calcium, a versatile second messenger of extracellular signals inside the cell, binds to a multitude of cytosolic Ca 2ϩ -binding proteins each of which can, in turn, regulate several effector proteins. The S100 protein family (18 different members) constitutes the largest group of Ca 2ϩ -binding proteins of the EF-hand type (1, 2). At least 13 S100 genes are clustered on human chromosome 1q21, leading to the designation S100A1 to S100A13 for the protein products of these genes (3). Their expression is cell-and tissue-specific, and different human diseases have been associated with deregulated expression (4, 5). S100 proteins are non-covalent homodimers with the notable exception of the heterodimeric S100A8-S100A9. Each monomer possesses 2 EF-hands as follows: a classical C-terminal EFhand with a canonical Ca 2ϩ -binding loop of 12 amino acids and a N-terminal EF-hand with a loop of 14 amino acids which is specific for S100 proteins. This structural difference likely is the reason for the large difference in the Ca 2ϩ affinities of the N-and C-terminal EF-hands. The affinities of S100 proteins for Ca 2ϩ are in general rather low with [Ca 2ϩ ] 0.5 1 values of 100 -300 mM (for review see Refs. 2 and 6), and in 6 out of 8 studied cases binding of Ca 2ϩ displays positive cooperativity. Zn 2ϩ binds to several S100 proteins; the Zn 2ϩ and Ca 2ϩ sites are distinct and can modify the affinity for Ca 2ϩ . S100B binds four Zn 2ϩ ions with concomitant 10-fold increases of the Ca 2ϩ affinity (7). High affinity binding of two Zn 2ϩ to the S100A12 dimer leads to induction of two high affinity Ca 2ϩ -binding sites (8). S100A3 binds eight Zn 2ϩ , but without effect on the affinity for Ca 2ϩ (9, 10). S100A2 binds four Zn 2ϩ with high affinity, in a manner antagonistic to Ca 2ϩ (11). Recently it was reported...

S100A13 Participates in the Release of Fibroblast Growth Factor 1 in Response to Heat Shock in Vitro

“…The finding that Myc-S100A13 expression is able to overcome the requirement for transcription and translation in the release of FGF1 is consistent with the observation that some members of the S100 gene family have been characterized not only as stress-induced genes (36 -39) but also as Cu 2ϩ -binding proteins (17,18,40,41). It is interesting to note that S100B was independently characterized from neural tissue as an inhibitor of Cu 2ϩ -mediated L-ascorbate oxidation (40).…”

“…However, several observations suggest that S100A13, unlike p40 Syt1, is able to facilitate the release of FGF1. Indeed, expression of Myc-S100A13 was able to overcome the inhibitory activity of actinomycin and cycloheximide on FGF1 release and was able to induce the release of Cys-free FGF1 in response to stress, suggesting a functional role for S100A13 in the FGF1 release pathway as a potential modifier of a stress-induced post-translational event.The finding that Myc-S100A13 expression is able to overcome the requirement for transcription and translation in the release of FGF1 is consistent with the observation that some members of the S100 gene family have been characterized not only as stress-induced genes (36 -39) but also as Cu 2ϩ -binding proteins (17,18,40,41). It is interesting to note that S100B was independently characterized from neural tissue as an inhibitor of Cu 2ϩ -mediated L-ascorbate oxidation (40).…”

S100A16, a Novel Calcium-binding Protein of the EF-hand Superfamily

“…Binding of Ca 2ϩ to S100B induces helix rearrangement within each subunit of the dimer, resulting in the exposure of two hydrophobic surfaces (one in each monomer) (38,40,41), which are involved in target protein recognition (36,37). Beside Ca 2ϩ , a number of S100 proteins can also bind Zn 2ϩ or Cu 2ϩ (42,43) that can influence the affinity of Ca 2ϩ binding (38). Thus, S100 proteins display variable transition metal-binding properties in agreement with their highly diversified and specialized functions.…”

Calbindin‐D28k

“…[22][23][24][25] The dilutions used for the S100 antibodies were 1/2000 for S100A1, 1/2000 for S100A3, 1/2000 for S100A4, 1/500 for S100A5, 1/10,000 for S100A6, and 1/10,000 for S100B.…”